Almost all RNAs are processed from a primary transcript to a mature RNA by a variety of enzymes, including RNases. During this process many mistakes are made and aberrant RNAs generated this way are rapidly degraded to maintain RNA homeostasis. The RNA exosome plays several critical roles in this. First, the nuclear exosome processes some RNAs from longer precursors. For example, it processes the 160 nt 5.8S rRNA from a 300 nt precursor. Second, the nuclear exosome completely degrades some RNAs that are byproducts of gene expression, including the 5' external transcribed spacer that is part of the rRNA precursor. Third, the nuclear exosome degrades aberrant RNAs that fail to complete proper processing. This includes the initiator tRNA that is not properly modified. Fourth, the exosome is also present in the cytoplasm, where it performs one of two general pathways of mRNA decay. Fifth, the cytoplasmic exosome appears especially important for degrading aberrant mRNAs, including those that lack a stop codon and those that lack a poly(A) tail. Probably related to the process of degrading aberrant mRNAs is that the exosome has an antiviral activity. The long term goal of this research is to understand the role of the RNA exosome in these processing and decay pathways. The core exosome consists of ten proteins, of which Rrp44p is the catalytic subunit. We and others have previously shown that Rrp44p contains a 3' to 5' exoribonucleolytic domain and an endonucleolytic domain. We propose research to better understand the contributions of the endonuclease activity to exosome function. While Rrp44p is the only catalytic subunit of the core exosome, the other nine core subunits are also essential for viability and for all exosome functions examined. We propose experiments aimed at better understanding the role of the other nine subunits. Although the purified exosome has RNase activity in vitro, its in vivo activity requires many cofactors. While some of these cofactors are required only for specific roles of the exosome, an RNA helicase of the Ski2-like family is required for all exosome functions. Ski2p itself is required for all cytoplasmic functions of the exosome, while the closely related Mtr4p is required for all nuclear functions of the exosome. Our proposed experiments seek to clarify how these helicases assist the exosome in its many function. PUBLIC HEALTH RELEVANCE: This research will use yeast as a model organism, but the exosome and its cofactors is highly conserved between yeast and humans. Thus the results should increase our understanding of the role of RNA processing and RNA degradation by the exosome in gene expression in humans as well.